Occurrence of multiple-antibiotic-resistant enteric bacteria in domestic sewage and oxidation lagoons.

The coliform bacterial population in the Grand Forks, N.Dak. sewage system was examined for multiple-antibiotic-resistant organisms over a 1-year period. Multiple-antibiotic-resistant coliforms were found to be common in the sewage, and their numbers remained fairly constant relative to the total coliform population throughout the year. Resistance to kanamycin, tetracycline, and ampicillin was found to be transferable at variable rates. Transfer rates were found to be temperature sensitive and were optimal at 35 degrees C. Although 75% of the multiple-antibiotic-resistant coliforms were capable of transferring resistance at some level, only 25% were capable of transferring resistance at rates greater than 10(-3) transconjugants per initial donor.     

Incidence of Infectious Drug Resistance Among Fecal Coliforms Isolated from Raw Sewage

Raw sewage was examined for the incidence of antibiotic-resistant coliforms present among both total and fecal coliforms. In both groups, it was found that approximately 3% of the coliform bacteria were resistant to two or more antibiotics. Of these organisms, 48% were capable of transferring all or part of their antibiotic resistance to an antibiotic-sensitive, F⁻, derivative of Escherichia coli K-12. Among the R factors identified, those conferring resistance to streptomycin-tetracycline, ampicillin-streptomycin-tetracycline, and ampicillin or ampicillin-streptomycin accounted for 23, 20, and 15%, respectively, of the total R factors detected. The data indicate a significant level of infectious drug resistance among the fecal coliforms of the urban population. The data indicate further that because of the high incidence of coliform bacteria found to be doubly resistant to streptomycin and tetracyline, the inclusion of these antibiotics in selective media used for routine total or fecal coliform counts may serve to identify domestic sources of pollution.     

Effect of UV light disinfection on antibiotic-resistant coliforms in wastewater effluents.

Total coliforms and total coliforms resistant to streptomycin, tetracycline, or chloramphenicol were isolated from filtered activated sludge effluents before and after UV light irradiation. Although the UV irradiation effectively disinfected the wastewater effluent, the percentage of the total surviving coliform population resistant to tetracycline or chloramphenicol was significantly higher than the percentage of the total coliform population resistant to those antibiotics before UV irradiation. This finding was attributed to the mechanism of R-factor-mediated resistance to tetracycline. No significant difference was noted for the percentage of the surviving total coliform population resistant to streptomycin before or after UV irradiation. Multiple drug resistance patterns of 300 total coliform isolates revealed that 82% were resistant to two or more antibiotics. Furthermore, 46% of these isolates were capable of transferring antibiotic resistance to a sensitive strain of Escherichia coli.     

R factors in coliform-fecal coliform sewage flora of the prairies and Northwest Territories of Canada.

Coliform and fecal coliform populations found in the raw sewages and final sewage effluents of the prairie provinces and the Northwest Territories were examined for antibiotic resistance and the possession of R factors. It was determined that 8.91% of the total coliform and 10.80% of the fecal coliform populations carried R factors. The following numbers of combinations of R determinants were found: 39 in the Escherichia coli population, 6 in the Citrobacter population, 20 in the Enterobacter populations, 10 in the Klebsiella populations, and 11 in the Aeromonas populations. The maximum number of R determinants transferable simultaneously was seven; organisms with R factors containing determinants for chloramphenicol usually contained determinants for ampicillin. Of the coliform and fecal coliform populations, 2 to 4% were resistant to chloramphenicol in some provinces, and from 17 to 30% of the populations were resistant to three or more antibiotics. It was calculated that coliforms containing R factors in the raw sewage reached population levels of 1.5 X 10(7)/100 ml, and fecal coliforms containing R factors reached population levels of 8.6 X 10(5) ml. Final effluent discharges to the receiving environment contained R factor-containing coliform and fecal coliform populations of 3.1 X 10(4)/100 ml and 5.8 X 10(2)/100 ml, respectively. The incidence of bacteria containing R factors in sewage appears to be increasing with time, and their removal from sewage before discharge to the receiving environment is desirable. Consideration of data on bacteria with R factors should be made in future water quality deliberations and in discharge regulations.     

Evaluation of m-T7 agar as a fecal coliform medium

m-T7 agar, designed to improve recoveries of injured total coliforms, was evaluated for its effectiveness as a fecal coliform medium. The time and temperature of preincubation were found to be crucial to the optimal recovery of fetal coliforms. Isolation rates for fecal coliforms on m-T7 agar from sewage effluents were the highest when plates were preincubated at 37 degrees C for 8 h before transfer to 44.5 degrees C for 12 h. The medium was found to produce consistently higher fecal coliform counts than all the other methods tested. Recoveries were 3.1 times greater than the standard m-FC method and 1.7 times greater than the two-layer enrichment, temperature acclimation procedure. Verification rates for fecal coliforms isolated on m-T7 agar averaged 89.0%, whereas verification rates for m-FC agar averaged only 82.8%. Both media isolated similar fecal coliform populations. The advantages of a single medium, highly effective for the isolation of both total and fecal coliforms, are discussed.     

Evaluation of m-T7 agar as a fecal coliform medium.

m-T7 agar, designed to improve recoveries of injured total coliforms, was evaluated for its effectiveness as a fecal coliform medium. The time and temperature of preincubation were found to be crucial to the optimal recovery of fetal coliforms. Isolation rates for fecal coliforms on m-T7 agar from sewage effluents were the highest when plates were preincubated at 37 degrees C for 8 h before transfer to 44.5 degrees C for 12 h. The medium was found to produce consistently higher fecal coliform counts than all the other methods tested. Recoveries were 3.1 times greater than the standard m-FC method and 1.7 times greater than the two-layer enrichment, temperature acclimation procedure. Verification rates for fecal coliforms isolated on m-T7 agar averaged 89.0%, whereas verification rates for m-FC agar averaged only 82.8%. Both media isolated similar fecal coliform populations. The advantages of a single medium, highly effective for the isolation of both total and fecal coliforms, are discussed.     

Influence of sewage treatment and urbanization on selection of multiple resistance in fecal coliform populations.

The fecal coliform populations found in the raw sewages and final sewage effluents of mechanical treatment plants, a long-term retention lagoon, shorter-term retention lagoons, a remote northern Canada river, and a heavily urbanized prairie river were examined for antibiotic resistance and the possession of R factors. It was determined that there was a decrease in the percentage of multiresistant fecal coliform populations in the mechanical sewage treatment plants and shorter-term retention lagoons; however, there was an increase in populations from the long-term retention lagoon. The percentage of the populations possessing transmissible R factors was constant in the mechanical treatment and shorter-term retention facilities; however, the ability to transmit was lost in 50% of the infective population of the long-term retention facility. A striking contrast was found between the populations of the remote northern Slave River and those of the urbanized Red River. Of the fecal coliforms in the Slave River, 7.1% were multiresistant, and only 0.79% possessed transmissible R factors. The Red River fecal coliform populations were 52.9% multiresistant, and 18.77% of the total population possessed transmissible R factors. The influence of urbanization and the type of sewage treatment have been shown to affect the selection and survival of multiresistant fecal coliforms and R+ fecal coliforms. Determination of other factors influencing the development and the survival of these populations is needed for rational wastewater management and water quality consideration.     

Antibiotic resistance patterns of fecal coliforms isolated from domestic sewage before and after treatment in an aerobic lagoon.

The resistance of 260 strains of fecal coliforms, isolated from raw domestic sewage and aerobic lagoon effluent, to ampicillin, aureomycin, chloromycetin, gentamicin, streptomycin, sulfadiazine, and tetracycline, was determined. Aerobic lagoon treatment produced a 20-fold reduction in the fecal coliform numbers. No statistically significant difference in antibiotic resistance was observed between the fecal coliforms found in raw sewage and in lagoon effluent despite a trend towards the loss of resistance in the latter. Antibiotic resistance, either single or multiple, did not contribute to, or detract from, bacterial survival in the aerobic lagoon. Of the isolates, 15% showed resistance to two or more antiobiotics.     

Sunlight inactivation of fecal indicator bacteria and bacteriophages from waste stabilization pond effluent in fresh and saline waters

Sunlight inactivation in fresh (river) water of fecal coliforms, enterococci, Escherichia coli, somatic coliphages, and F-RNA phages from waste stabilization pond (WSP) effluent was compared. Ten experiments were conducted outdoors in 300-liter chambers, held at 14C (mean river water temperature). Sunlight inactivation (k(S)) rates, as a function of cumulative global solar radiation (insolation), were all more than 10 times higher than the corresponding dark inactivation (k(D)) rates in enclosed (control) chambers. The overall k(S) ranking (from greatest to least inactivation) was as follows: enterococci > fecal coliforms greater-than-or-equal E. coli > somatic coliphages > F-RNA phages. In winter, fecal coliform and enterococci inactivation rates were similar but, in summer, enterococci were inactivated far more rapidly. In four experiments that included freshwater-raw sewage mixtures, enterococci survived longer than fecal coliforms (a pattern opposite to that observed with the WSP effluent), but there was little difference in phage inactivation between effluents. In two experiments which included simulated estuarine water and seawater, sunlight inactivation of all of the indicators increased with increasing salinity. Inactivation rates in freshwater, as seen under different optical filters, decreased with the increase in the spectral cutoff (50% light transmission) wavelength. The enterococci and F-RNA phages were inactivated by a wide range of wavelengths, suggesting photooxidative damage. Inactivation of fecal coliforms and somatic coliphages was mainly by shorter (UV-B) wavelengths, a result consistent with photobiological damage. Fecal coliform repair mechanisms appear to be activated in WSPs, and the surviving cells exhibit greater sunlight resistance in natural waters than those from raw sewage. In contrast, enterococci appear to suffer photooxidative damage in WSPs, rendering them susceptible to further photooxidative damage after discharge. This suggests that they are unsuitable as indicators of WSP effluent discharges to natural waters. Although somatic coliphages are more sunlight resistant than the other indicators in seawater, F-RNA phages are the most resistant in freshwater, where they may thus better represent enteric virus survival.     

Automated electrical impedance technique for rapid enumeration of fecal coliforms in effluents from sewage treatment plants.

Fecal coliforms growing in a selective lactose-based broth medium at 44.5 degrees C generate a change in the electrical impedance of the culture relative to a sterile control when populations reach 10(6) to 10(7) per ml. The ratio of these changes was measured automatically, and the data were processed by computer. A linear relation was found between the log10 of the number of fecal coliforms in an inoculum and the time required for an electrical impedance ratio signal to be detected. Pure culture inocula consisting of 100 fecal coliforms in log phase or stationary phase were detected in 6.5 and 7.7 h, respectively. Standard curves of log10 fecal coliforms in wastewater inocula versus detection time, based on samples collected at a sewage treatment plant over a 4-month period, were found to vary from one another with time. Nevertheless, detection times were rapid and ranged from 5.8 to 7.9 h for 200 fecal coliforms to 8.7 to 11.4 h for 1 fecal coliform. Variations in detection times for a given number of fecal coliforms were also found among sewage treatment plants. A strategy is proposed which takes these variations into account and allows for rapid, automated enumeration of fecal coliforms in wastewater by the electrical impedance ratio technique.     

Automated electrical impedance technique for rapid enumeration of fecal coliforms in effluents from sewage treatment plants.

Fecal coliforms growing in a selective lactose-based broth medium at 44.5 degrees C generate a change in the electrical impedance of the culture relative to a sterile control when populations reach 10(6) to 10(7) per ml. The ratio of these changes was measured automatically, and the data were processed by computer. A linear relation was found between the log10 of the number of fecal coliforms in an inoculum and the time required for an electrical impedance ratio signal to be detected. Pure culture inocula consisting of 100 fecal coliforms in log phase or stationary phase were detected in 6.5 and 7.7 h, respectively. Standard curves of log10 fecal coliforms in wastewater inocula versus detection time, based on samples collected at a sewage treatment plant over a 4-month period, were found to vary from one another with time. Nevertheless, detection times were rapid and ranged from 5.8 to 7.9 h for 200 fecal coliforms to 8.7 to 11.4 h for 1 fecal coliform. Variations in detection times for a given number of fecal coliforms were also found among sewage treatment plants. A strategy is proposed which takes these variations into account and allows for rapid, automated enumeration of fecal coliforms in wastewater by the electrical impedance ratio technique.     

Use of beta-D-galactosidase and beta-D-glucuronidase activities for quantitative detection of total and fecal coliforms in wastewater

Two enzymatic methods based on the measurement of the beta-D-galactosidase activity of total coliforms and the beta-D-glucuronidase activity of Escherichia coli were used to assess coliform levels in wastewater alongside traditional culture-based techniques, which can be biased by the aggregation of bacteria or their attachment to particles. Enzymatic methods were precise (i.e., coefficients of variation were 9%-15%), rapid (response in 20 min), and correlated well (in log units) with traditional techniques for raw and treated sewage (r2 > 0.75). They were used for rapid assessment of coliform removal efficiency in two different wastewater treatments. These methods could be useful for the estimation of the abundance of coliforms in domestic sewage and their removal by wastewater treatment processes.     

Effects of waste water treatment on the species composition and Antibiotic resistance of coliform bacteria

Coliform bacteria from raw and treated sewage in a mechanical-biological treatment plant were examined for resistance to four to six antibiotics, and randomly selected isolates were identified. The results indicate that certain coliform species are eliminated at lower rates than others by the treatment.Klebsiella sp. became more frequent, whereas the proportion ofEscherichia coli diminished. A comparison of raw and treated sewage clearly reveals that the resistance patterns are transient characteristics, and resistance percentages may either decrease or increase following passage through the treatment plant. Mating of multiresistant isolates withE. coli K12 donor confirmed that some of the resistance traits are borne on conjugative plasmids. Multiple antibiotic resistance (MAR) indexing revealed that resistance levels ofE. coli decreased during waste water treatment, but increased in total coliforms.     

A note on antibiotic resistance in the bacterial flora of raw sewage and sewage-polluted River Tigris in Mosul, Iraq

Polluted water samples collected from the River Tigris in the vicinity of a raw sewage outfall were examined for the incidence of antibiotic resistance among coliform bacteria on three occasions during 1983. Eighty percent or more of the coliform bacteria were resistant to one or more antibiotics. At the same time, raw sewage samples were examined for the incidence of antibiotic-resistant bacteria, and Escherichia coli, Pseudomonas spp. and Staphylococcus spp. were selected for sensitivity testing. Collectively, more than 90% of the 480 strains of the three organisms were resistant to one or more antibiotics. The minimal inhibitory concentration (MIC) of ampicillin for twenty-nine strains including coliforms, E. coli, Klebsiella sp., Serratia sp., Ps. aeruginosa, Pseudomonas sp., Micrococcus sp., Staph. aureus, Streptococcus faecalis and Bacillus sp. from raw sewage and polluted River Tigris water was determined and that for Ps. aeruginosa was 250 micrograms/ml. The high incidence of antibiotic-resistant bacteria in natural waters could be related to the widespread use of antibiotics in this locality.     

Sunlight Inactivation of Fecal Indicator Bacteria and Bacteriophages from Waste Stabilization Pond Effluent in Fresh and Saline Waters

Sunlight inactivation in fresh (river) water of fecal coliforms, enterococci, Escherichia coli, somatic coliphages, and F-RNA phages from waste stabilization pond (WSP) effluent was compared. Ten experiments were conducted outdoors in 300-liter chambers, held at 14°C (mean river water temperature). Sunlight inactivation (k_S) rates, as a function of cumulative global solar radiation (insolation), were all more than 10 times higher than the corresponding dark inactivation (k_D) rates in enclosed (control) chambers. The overall k_S ranking (from greatest to least inactivation) was as follows: enterococci > fecal coliforms ≥ E. coli > somatic coliphages > F-RNA phages. In winter, fecal coliform and enterococci inactivation rates were similar but, in summer, enterococci were inactivated far more rapidly. In four experiments that included freshwater-raw sewage mixtures, enterococci survived longer than fecal coliforms (a pattern opposite to that observed with the WSP effluent), but there was little difference in phage inactivation between effluents. In two experiments which included simulated estuarine water and seawater, sunlight inactivation of all of the indicators increased with increasing salinity. Inactivation rates in freshwater, as seen under different optical filters, decreased with the increase in the spectral cutoff (50% light transmission) wavelength. The enterococci and F-RNA phages were inactivated by a wide range of wavelengths, suggesting photooxidative damage. Inactivation of fecal coliforms and somatic coliphages was mainly by shorter (UV-B) wavelengths, a result consistent with photobiological damage. Fecal coliform repair mechanisms appear to be activated in WSPs, and the surviving cells exhibit greater sunlight resistance in natural waters than those from raw sewage. In contrast, enterococci appear to suffer photooxidative damage in WSPs, rendering them susceptible to further photooxidative damage after discharge. This suggests that they are unsuitable as indicators of WSP effluent discharges to natural waters. Although somatic coliphages are more sunlight resistant than the other indicators in seawater, F-RNA phages are the most resistant in freshwater, where they may thus better represent enteric virus survival.     

Modification of M-FC medium by eliminating rosolic acid.

Eliminating rosolic acid from M-FC medium improves the MFC procedure by allowing higher fecal coliform colony recoveries with greater ease in counting. Samples of unchlorinated and chlorinated domestic sewage, creek, lake, and river water were analyzed for fecal coliforms by standard procedures. Results of 200 comparisons of fecal coliform counts on M-FC medium without and with rosolic acid showed that higher counts were obtained 71% of the time when rosolic acid was excluded without an overgrowth of background colonies. Results from analyzing chlorinated sewage showed that eliminating rosolic acid improved the recovery of fecal coliform bacteria by 49%. A total of 1,675 blue colonies and 766 nonblue colonies were verified. Of the 1,675 blue colonies, 1,566 were confirmed as fecal coliform bacteria, for a verification of 93.5%. The percent verification of nonblue colonies as noncoliform bacteria was 84.2% (644/766).     

Modification of M-FC medium by eliminating rosolic acid.

Eliminating rosolic acid from M-FC medium improves the MFC procedure by allowing higher fecal coliform colony recoveries with greater ease in counting. Samples of unchlorinated and chlorinated domestic sewage, creek, lake, and river water were analyzed for fecal coliforms by standard procedures. Results of 200 comparisons of fecal coliform counts on M-FC medium without and with rosolic acid showed that higher counts were obtained 71% of the time when rosolic acid was excluded without an overgrowth of background colonies. Results from analyzing chlorinated sewage showed that eliminating rosolic acid improved the recovery of fecal coliform bacteria by 49%. A total of 1,675 blue colonies and 766 nonblue colonies were verified. Of the 1,675 blue colonies, 1,566 were confirmed as fecal coliform bacteria, for a verification of 93.5%. The percent verification of nonblue colonies as noncoliform bacteria was 84.2% (644/766).     

Repair detection procedure for enumeration of fecal coliforms and enterococci from seafoods and marine environments.

The repair detection procedure of Speck et al. (Appl. Microbiol. 29:549-550, 1975) was adapted for the enumeration of coliforms, fecal coliforms, and enterococci in seafood and environmental samples. Samples were pour plated with Trypticase soy agar, followed by a 1- to 2-h incubation to effect repair; the plates were then overlaid with the selective medium and incubated. Violet red bile agar and an incubation temperature of 45 degrees C were used as the selective conditions for fecal coliforms, and KF streptococcal agar was used for the enumeration of enterococci. The method was more efficient than the standard most-probable-number method for fecal coliform enumeration and also allowed enumeration of the injured cells, which might have remained undetected when selective medium in the most-probable-number method was used. The repair detection method effectively recovered the injured portion of the population of enterococci capable of growing on KF streptococcal agar. The repair enumeration method was not suitable for coliforms in marine samples because associative marine bacteria mimicked coliforms in violet red bile agar plates incubated at 35 degrees C. The marine bacteria did not grow at 45 degrees C and therefore did not interfere with fecal coliform enumeration.     

Repair detection procedure for enumeration of fecal coliforms and enterococci from seafoods and marine environments.

The repair detection procedure of Speck et al. (Appl. Microbiol. 29:549-550, 1975) was adapted for the enumeration of coliforms, fecal coliforms, and enterococci in seafood and environmental samples. Samples were pour plated with Trypticase soy agar, followed by a 1- to 2-h incubation to effect repair; the plates were then overlaid with the selective medium and incubated. Violet red bile agar and an incubation temperature of 45 degrees C were used as the selective conditions for fecal coliforms, and KF streptococcal agar was used for the enumeration of enterococci. The method was more efficient than the standard most-probable-number method for fecal coliform enumeration and also allowed enumeration of the injured cells, which might have remained undetected when selective medium in the most-probable-number method was used. The repair detection method effectively recovered the injured portion of the population of enterococci capable of growing on KF streptococcal agar. The repair enumeration method was not suitable for coliforms in marine samples because associative marine bacteria mimicked coliforms in violet red bile agar plates incubated at 35 degrees C. The marine bacteria did not grow at 45 degrees C and therefore did not interfere with fecal coliform enumeration.     

Influence of seasonal environmental variables on the distribution of presumptive fecal Coliforms around an Antarctic research station

Factors affecting fecal microorganism survival and distribution in the Antarctic marine environment include solar radiation, water salinity, temperature, sea ice conditions, and fecal input by humans and local wildlife populations. This study assessed the influence of these factors on the distribution of presumptive fecal coliforms around Rothera Point, Adelaide Island, Antarctic Peninsula during the austral summer and winter of February 1999 to September 1999. Each factor had a different degree of influence depending on the time of year. In summer (February), although the station population was high, presumptive fecal coliform concentrations were low, probably due to the biologically damaging effects of solar radiation. However, summer algal blooms reduced penetration of solar radiation into the water column. By early winter (April), fecal coliform concentrations were high, due to increased fecal input by migrant wildlife, while solar radiation doses were low. By late winter (September), fecal coliform concentrations were high near the station sewage outfall, as sea ice formation limited solar radiation penetration into the sea and prevented wind-driven water circulation near the outfall. During this study, environmental factors masked the effect of station population numbers on sewage plume size. If sewage production increases throughout the Antarctic, environmental factors may become less significant and effective sewage waste management will become increasingly important. These findings highlight the need for year-round monitoring of fecal coliform distribution in Antarctic waters near research stations to produce realistic evaluations of sewage pollution persistence and dispersal.